1. Molecular Therapy - Nucleic Acids
miR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis 
Li Zhou, Shunai Liu, Ming Han, Yanhua Ma, Shenghu Feng, Jing Zhao, Hongping Lu, Xiaoxue Yuan, Jun Cheng 
Molecular Therapy - Nucleic Acids 
Volume 10, Pages (March 2018)
DOI: /j.omtn
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
miR-185 Is Downregulated in the Plasma of Patients with HBV-Related Liver Fibrosis
The expression of miRNAs in the plasma of patients (n = 10) and control (n = 8) groups was detected by Illumina HiSeq sequencing. (A) Levels of miR-185-5p were significantly lower in the patient group than in the healthy group. (B) The expression of miR-185-3p was downregulated compared with the control group. (*p < , **p < ).
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
miR-185 Is Abundant in LX-2 Cells but Downregulated in Activated LX-2 Cells
The human hepatic stellate cell (HSC) cell line LX-2 was treated with 5.0 ng/mL transforming growth factor β-1 (TGF-β1) for 0, 3, 6, 12, or 24 hr or with 2.5, 5.0, or 7.5 ng/mL TGF-β1 for 24.0 hr. (A) mRNA expression of key genes involved in the activation of HSC α-SMA, COL1A1, COL1A2, and COL3A1 was analyzed by real-time PCR. All genes increased in a time-dependent manner. mRNA levels of Ras homolog enriched in brain (RHEB) and rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR) were assessed by real-time PCR. When HSCs were activated by TGF-β1 with a different dose (B) or time (C), RHEB and RICTOR were upregulated in mRNA levels. (D) Protein expressions of α-SMA, COL I, COL III, RHEB, and RICTOR was determined by western blotting; GAPDH or β-actin was used as a loading control. When HSCs were activated, all genes were upregulated in protein levels. miR-185-5p expression was reduced in activated HSCs in a dose-dependent manner (E) or time-dependent manner (F) examined by real-time PCR (n = 3, *p < , **p < 0.01). Data represent mean ± SEM.
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
miR-185-Suppressed HSC Activation and Basal/TGF-β1-Induced Fibrous ECM Production of LX-2 Cells
LX-2 cells were seeded into six-well plates at a density of 1.0 × 106 cells per well, and cells were transiently transfected overnight with miR-185 mimics (miR-185) or negative mimic controls (miR-NC). (A and B) After 48 hr, (A) real-time qPCR was performed to analyze the mRNA levels, and (B) western blotting was performed to analyze the protein levels of liver fibrogenesis-related genes. miR-185 mimics downregulated mRNA and protein expression levels of COL I, COL III, and α-SMA. (C) Western blotting data were quantified using the ImageJ software. (D) To detect the effects of miR-185 on TGF-β1-induced profibrogenic gene expression, LX-2 cells were transfected with miR-185 mimics for 24 hr and then replaced with new culture medium treated with or without 5.0 ng/mL TGF-β1 for another 24 hr. α-SMA, COL I, and COL III protein levels were detected by western blotting. miR-185 can repress phenotypes associated with TGF-β1-induced transition of resident fibroblasts. The results are representative of three independent experiments and are presented as mean ± SEM (n = 3, *p < , **p < ).
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Knockdown of Endogenous miR-185 Promoted Basal/TGF-β1-Induced Liver Fibrosis
LX-2 cells were transfected with inhibitor-NC or miR-185 inhibitor for 48 hr; inhibition of miR-185 in LX-2 cells induced liver fibrogenesis-related gene transcription (A) and translocation (B). (C) Western blot data were quantified. (D) TGF-β1-induced liver fibrosis, and miR-185 inhibitors (anti-miR-185) upregulated protein expression levels of COL I, COL III, and α-SMA after TGF-β1 further induced HSC activation. Three independent experiments gave similar results (mean ± SEM, n = 3, *p < , **p < 0 0.01).
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
miR-185 Directly Binds to the 3′ UTRs of the RHEB and RICTOR mRNAs
pmirGLO vectors containing either the wild-type (WT) or mutant (mut) binding sites of miR-185-5p in RHEB (A) and RICTOR (B) mRNAs were constructed to conduct luciferase reporter assays as described previously. Dual-luciferase reporter assays were performed to verify binding between miR-185-5p and RHEB and RICTOR mRNA. The HSC line LX-2 was co-transfected with a pmirGLO vector containing either the WT or mut target sites plus either the miR-185-5p mimics or the mimic control oligonucleotide. Results of relative luciferase activity are shown as the mean ± SEM obtained from triplicate experiments. (C–E and G) RHEB and RICTOR mRNA and protein levels were analyzed by real-time qPCR (C and D) and western blotting (E and G), respectively. The mRNA and protein levels of RHEB and RICTOR were downregulated in LX-2 cells transfected with miR-185 mimics but upregulated when transfected with miR-185 inhibitors. (F and H) Western blotting data were quantified using the ImageJ software. Data shown represent the means ± SEM of independent experiments (unpaired two-sample Student’s t test, n = 3, **p < , *p < ).
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
RHEB and RICTOR Mediated the Effects of miR-185 on LX-2 Cell Activation
Quantitative real-time PCR determined α-SMA, COL1A1, COL1A2, and COL3A1 mRNA expression in LX-2 cells transfected with si-RHEB (A) and si-RICTOR (B). The reduction of RHEB and RICTOR expression also blocked HSC activation at the mRNA level. Three siRNA oligos interfering with the same specific target genes (RHEB/RICTOR) to produce a similar effect were designed by GenePharma. Western blotting was performed to confirm the knockdown of RHEB (C) and RICTOR (D) expression and fibrogenesis-related gene protein expression levels in LX-2 cells after transfection with si-RHEB and si-RICTOR. The first si-RHEB seems to be the most effective si-RNA oligo, and three si-RICTOR oligos are all effective. The reduction of RHEB and RICTOR expression also blocked HSC activation at the protein level.
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

8. Figure 7
CCl4-Induced Liver Fibrosis in Mice Downregulates miR-185 and Upregulates RHEB and RICTOR
(A) The histopathological changes in livers 1 month after injection of CCl4 are shown by H&E staining, Masson’s trichrome staining, and Sirius red staining of sections from two representative livers. (B) Real-time qPCR analysis for α-SMA, COL1A1, COL1A2, COL3A1, RHEB, and RICTOR in two groups. mRNA levels increased in all genes. All results of relative expression values are shown as the mean ± SEM. (C) Western blotting analysis for α-SMA, fibronectin (FN), TGFR1, RHEB, RICTOR, and GAPDH in livers of representative mice from each group. The protein levels were upregulated in the CCl4 group. α-SMA and collagen are obviously increased, and the changes in RHEB and RICTOR levels are modest. (D) Data represent one of three experiments with similar results. Western blotting data were quantified using the ImageJ software. (E) Immunohistochemistry for α-SMA, RHEB, and RICTOR in liver sections from representative NC and CCl4 livers. In the model group, protein levels of α-SMA, RHEB, and RICTOR increased. (F) Real-time qPCR measured miR-185-5p expression in livers from NC and CCl4-treated mice. miR-185-5p expression was reduced in the CCl4 group.
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions

9. Figure 8
Schematic
TGF-β1-induced activated HSCs also downregulate miR-185 concomitant with increased expression of RHEB and RICTOR. miR-185 prevents liver fibrogenesis by inhibiting HSC activation through inhibiting RHEB and RICTOR.
Molecular Therapy - Nucleic Acids  , DOI: ( /j.omtn )
Copyright © 2017 The Authors Terms and Conditions